Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing

Area of Science:

• Biotechnology
• Molecular Biology
• Drug Discovery

Background:

• Genetically-encoded (GE) libraries are crucial for identifying novel ligands.
• Understanding biases within these libraries is essential for successful ligand discovery.
• The M13KE phage display platform is widely used for constructing GE libraries.

Purpose of the Study:

• To investigate biases in GE libraries of tetrapeptides displayed on the M13KE platform.
• To analyze the impact of different library construction stages (synthetic DNA, ligated vector, naive library, chemically modified library) on diversity.
• To identify amino acid censorship arising from phage display and chemical post-translational modification (cPTM).

Main Methods:

• Construction of GE libraries using trinucleotide cassette synthesis and NNK randomization.
• Chemical modification including aldehyde ligation and cysteine macrocyclization.
• Paired-end deep sequencing to characterize library composition at different stages.

Main Results:

• A significant drop in diversity was observed from ligated vector to naive library ({L}→{N}).
• Libraries expressed at the N-terminus of phage protein pIII censored Arg and Lys.
• Expression between pIII domains N1 and N2 reduced Arg/Lys censorship but introduced bias towards Gly and Ser.
• Chemical post-translational modification (cPTM) by aldehyde ligation and cysteine macrocyclization censored Ser and Phe.

Conclusions:

• Phage display and cPTM introduce distinct biases affecting GE library composition.
• Specific expression strategies and cPTM methods can be optimized to mitigate identified biases.
• The analytical approach can be applied to evaluate and enhance diversity in new display platforms and cPTM strategies.